dna vaccines, methods and protocols

Cancer gene therapy using plasmid DNA: purification and DNA for human clinical trials... Such contamina-tion includes components used in the isolation process and coming from theorganism

From: Methods in Molecular Medicine, vol 29, DNA Vaccines: Methods and Protocols

Edited by: D B Lowrie and R G Whalen Humana Press Inc., Totowa, NJ

large scale and still result in compromised purity (1,2) The success of DNA vaccines in animal models and the initiation of human trials (3,4) has led to a

need to increase the level of supercoiled plasmid purity as well as the ology utilized to produce these plasmids at large scale Several parameters ofthe purification process need to be addressed:

method-• The ability to prepare supercoiled plasmid at purity levels acceptable for cal material

clini-• The ability to prepare clinical grade supercoiled plasmid that will be scalable inorder to produce gram quantities of product

• The ability to prepare clinical grade supercoiled plasmid in accordance withcGMP principles

• The ability to develop validated assays to assess purity, yield, and tion levels

contamina-Challenges to the successful development of a purification process can bedivided into biological and practical The biological challenge arises from thespectrum of biomolecules that must be purified away from the supercoiled plas-

mid product ( Table 1) Additionally, the spectrum of nucleic acid

contami-nants and plasmid isoforms within that spectrum, as shown in Table 2, must be

removed The removal of the relaxed DNA, DNA catenanes as well as

endo-toxins (5,6) are a particular problem requiring additional steps in the process.

The practical challenge arises because the purification process that is oped must produce highly pure product at high yield and must be reproducible,

devel-scalable and economical ( see Note 1).

We describe a new purification process that has been used to generate cal material using a proprietary non-porous polymer resin, PolyFlo ®, whichuses principles of ion-pair reverse-phase chromatography to achieve separa-tion based on size and charge density The process can be performed usingeither acetonitrile (ACN) or ethanol (EtOH) Simultaneous removal of con-taminating endotoxins, chromosomal DNA, RNA, proteins, and plasmid iso-forms during purification is a unique advantage of this resin This process meets

clini-the challenges for purity, yield, reproducibility, and scalability (1).

2 Materials

2.1 Crude Starting Material

The preparation of crude starting material from biomass is typically

per-formed using acid/base extraction (7) This classic alkaline-lysis process

pro-vides material significantly reduced in protein, lipid and chromosomal DNA.Newer protocols have been adopted to improve the initial purity even further,including the use of temperature shift during fermentation (8) or the addition

of a second acetate precipitation (NH4Ac) to reduce the RNA burden (9) We

describe two purification methods: an ACN process using starting material in

which minimal efforts have been made to reduce the RNA burden, and (2) an

EtOH process in which the starting material has been reduced for RNA byanion-exchange chromatography and/or diafiltration The protocols described

do not incorporate the use of RNase ( see Note 2).

2.2 ACN Purification Materials

1 Glass borosilicate chromatography column packed with PolyFlo resin (see

Monomer supercoiledNicked

LinearDimersCatenanes

3 1.0 M TEAA (triethylamine acetate) pH 7.0.

4 0.5 M KPO4 pH 7.0

5 1.0 M TBAP (tetrabutylammonium phosphate); Aldrich Chemicals (Milwaukee, WI) no.# 26, 810-0; 1 M in H2O)

6 100% Acetonitrile (ACN, American Chemical Society (ACS) grade or equivalent)

7 TES (20 mM Tris-HCl pH 8.0, 1.0 mM EDTA, 5.0 mM NaCl).

8 Equilibration buffer: 0.1 M TEAA pH 7.2, 6% ACN.

9 Wash buffer I: TES, 5% ACN

10 Wash buffer II: 0.1 M KPO4, 2.0 mM TBAP, 5% ACN.

11 Wash buffer III: 0.1M KPO4, 2.0 mM TBAP, 15% ACN.

12 Elution buffer: 0.1 M KPO4, 2.0 mM TBAP, 25% ACN.

13 Sanitization buffer: 0.5 N NaOH

2.3 Ethanol Purification Materials

1 Glass borosilicate chromatography column packed with PolyFlo resin

2 RNA-reduced plasmid sample

3 0.5 M KPO4 pH 7.0

4 1.0 M TBAP (tetrabutylammonium phosphate; Aldrich Chemicals # 26, 810-0;

1 M in H2O)

5 Ethanol (EtOH, ACS grade or equivalent)

6 TES (0.02 M Tris-HCl pH 8.0, 1.0 mM EDTA, 5.0 mM NaCl).

7 Equilibration buffer: 0.1 M KPO4 pH 7.0, 2.0 mM TBAP, 1% ethanol.

8 Wash buffer I: TES, 7% ethanol

9 Wash buffer II: 0.1 M KPO4, 2.0 mM TBAP, 5% ethanol.

10 Elution buffer: 0.1 M KPO4, 2.0 mM TBAP, 25% ethanol.

11 Sanitization buffer: 0.5 N NaOH

2.4 Post-Purification Materials

Millipore Pellicon II (Bedford, MA) or A/G Technology (Needham, MA)diafiltration/ultrafiltration technologies are applicable for buffer exchange andconcentration

2 Prepare sample by diluting 1/5 with TES and adjusting to 0.1 M TEAA using 1 M

TEAA stock solution Sample load should be no more than 0.5 mg/mL of resin(load concentration is based on total A260nm)

3 Load sample and wash with equilibration buffer until the monitor returns tobaseline (~2 column volumes) Collect wash

4 Wash with ~3 column volumes of wash buffer I (TES, 5% ACN) Make suremonitor returns to baseline Collect wash.

5 Wash with ~3 column volumes of wash buffer II (0.1 M KPO4, 2.0 mM TBAP,

5% ACN)

6 Wash with ~3 column volumes of wash buffer III (0.1 M KPO4, 2.0 mM TBAP,

15% ACN) until the monitor returns to baseline Collect wash

7 Elute product with a 10-column volumes linear gradient from 0.1 M KPO4, 2.0

mM TBAP, 15% ACN to 0.1 M KPO4, 2.0 mM TBAP, 25% ACN Collect elution

fractions This is the purified product

8 Clean column by running 2 column volumes of sanitization buffer (0.5 N NaOH)

Turn pump off and let sit in 0.5 N NaOH for 1 h Re-equilibrate column with ≥3column volumes of equilibration buffer Monitor pH to assure that all the NaOHhas been removed

9 Purified sample from step 8 may be processed through concentration and/or

buffer exchange steps It is recommended to diafilter against 0.5 M Na-acetate

pH 7.8 to remove residual TBAP

3.2 ACN Protocol Results

The results of the ACN protocol are shown in Fig 2 No difference in the

purity of the product is seen using starting material representing 1 g or 100 g ofbiomass The RNA is eliminated Despite the significant quantities of relaxedDNA, >50% of total plasmid, this contaminant is removed in the wash step.The final purity is >90%

2 Prepare sample for loading by adjusting to 2 mM TBAP and 1% ethanol Sample

load should be no more than 0.5 mg/mL of resin (load concentration is based ontotal A260nm) Sample should be <0.5 M NaCl.

3 Load sample and wash with equilibration buffer until the monitor returns tobaseline Collect flow through

4 Wash with ~3 column volumes of wash buffer I (TES, 6% ethanol) Make suremonitor returns to baseline Collect wash

5 Wash with ~3 column volumes of wash buffer II (0.1 M KPO4, 2.0 mM TBAP,

5% ethanol)

6 Elute product with a 10-column volumes linear gradient from 0.1 M KPO4, 2.0 mM TBAP, 5% Ethanol to 0.1 M KPO4, 2.0 mM TBAP, 25% ethanol Collect elution

fractions This is the purified product

7 Clean column by running 2 column volumes of sanitization buffer (0.5 N NaOH).Turn pump off and let sit in 0.5 N NaOH for 1 h Re-equilibrate column with ≥3column volumes of equilibration buffer Monitor pH to assure that all the NaOHhas been removed

Fig 2 PolyFlo chromatography of plasmid using ACN process (A)

Chromato-graphic tracing for application of nucleic acid sample extracted from 1.0 g E coli

cells (B,C) 1% Agarose gel analysis of resolved peaks from 1.0 g biomass (B) and

100 g biomass (C) Lane 1 = 1 µg nucleic acid sample; lanes 4 and 5 = TES and ACNwash; lanes 6 and 7 = 15% (v/v) ACN/TBAP wash; lanes 8 and 9 = 15-25% (v/v) ACN

gradient; and lane 10 = 50% ACN strip Reprinted from (1).

8 Purified sample from step 7 may be processed through concentration and/or

buffer exchange steps It is recommended to diafilter against 0.5 M Na-acetate

pH 7.8 to remove residual TBAP

3.4 Ethanol Protocol Results

The results of the Ethanol protocol are shown in Fig 3 The residual RNA is

eliminated Despite the significant quantities of relaxed DNA, >60% of mid, this contaminant is removed in the wash step The final purity is >95%

plas-4 Notes

4.1 Organic Solvent

The choice of organic solvent for chromatography is predicated on theamount of contaminating RNA In general, if the RNA burden is less than 50%,the ethanol process may be employed This can be accomplished through anion-exchange chromatography, diafiltration or RNase treatment A rigorous test ofthe amount of contaminating RNA below which the ethanol process can beused has not been performed If RNA reduction is achieved by RNase diges-tion, the sample must be diafiltered or dialyzed to remove excess ribonucle-otides prior to PolyFlo chromatography

Fig 3 PolyFlo Chromatography of plasmid using EtOH process Crude lysate wasreduced for RNA by diafiltration against 10–20 vol TES using a Millipore XL mem-brane (100,000 MWCO) prior to application onto a 1 × 4 cm PolyFlo column (A) Chromatographic tracing at 254 nm; (B) 1% Agarose gel of resolved peaks Lane 1 =

starting material; lane 2 = 1% EtOH flow-through peak; lane 3 = 6% EtOH wash peak;and lane 4 = 5–25% EtOH gradient

(10) While many methods can be used to analyze plasmid DNA, it is only

recently that these methods have been applied to plasmid DNA as a potential

pharmaceutical product (11) Table 3 describes some of the target

specifica-tions and methods used within the industry

4.4 Multiple Chromatography Runs

PolyFlo is a chemically inert polymer that withstands rigorous sanitizationprocedures which allows for multiple runs One hundred consecutive applica-tions of crude plasmid with no change in purity or contamination levels have

Fig 4 Endotoxin binding to a PolyFlo column (1 × 4 cm) (A) Total endotoxin

units (EU) were determined in the flow-through (solid squares) and gradient elution(open circles) after loading sample buffer was spiked with increasing levels of endo-

toxin (B) Analysis of endotoxin levels in purified plasmid preparations at defined

intervals during 100 consecutive applications of a single PolyFlo chromatography

col-umn Reprinted from (1).

been documented (1) The resin can be sanitized to remove any residual nucleic

acid, protein, lipid and endotoxin by exposure to 0.5 N NaOH alone or in

com-bination with 0.1 M HCl.

4.5 Potential Interferences

PolyFlo resin is sensitive to detergents but not chaotropes or salts The use

of PEG in the lysis process does not affect PolyFlo performance (1) However,

detergents such as SDS, in concentrations >0.005% prevent binding to the resinand should be avoided

4.6 Process Optimization

As with all chromatographic procedures, there are several key steps that willaffect results Using PolyFlo in the chromatography of plasmids, the key ele-ments are the organic solvent concentration in the loading buffer and the col-umn wash steps For example, if the load concentration of organic is too high,product will be lost in the flow-through fraction If the load concentration istoo low, then the RNA will bind and will not be eliminated in the flow-through

As a consequence, trace RNA levels may be seen throughout the phy The same considerations can be applied to the wash concentration

chromatogra-References

1 Green, A P., Prior, G P., Helveston, N M., Taittinger, B E., Liu, X.-F., andThompson, J A (1997) Preparative purification of supercoiled plasmid DNA for

therapeutic applications BioPharm 10, 52–62.

2 Horn, N A., Meek, J A., Budahazi, G., and Marquet, M (1995) Cancer gene

therapy using plasmid DNA: purification and DNA for human clinical trials Hum.

Genomic DNA <1% Slot-blot hybridization

3 Ulmer, J B., Donnelly, J J., and Liu, M (1996) Toward the development of

DNA vaccines Curr Opin Biotechnol 7, 653–658.

4 Donnelly, J J., Ulmer , J B., Shiver, J W., and Liu, M (1997) DNA vaccines

Ann Rev Immunol 15, 617–648.

5 Weber, M., Möller, K., Welzeck, M., and Schoor, J (1995) Effects of

lipopolysac-charide on transfection efficiency in eukaryotic cell BioTechniques 19, 930–940.

6 Wicks, I P., Howell, M L., Hancock, T., Kohsaka, H., Olee, T -W., and Carson,

D (1995) Bacterial lipopolysaccharide copurifies with plasmid DNA:

implica-tions for animal models and human gene therapy Hum Gene Ther 6, 317–323.

7 Birnbaum, H C and Doly, J (1979) A rapid alkaline extraction procedure for

screening recombinant plasmid DNA Nucleic Acids Res 7, 1513–1523.

8 Lahijani, R., Hulley, G., Soriano, G., Horn, N., and Marquet, M (1996) yield production of pBR322-derived plasmids intended for human gene therapy

High-by employing a temperature-controllable point mutation Hum Gene Ther 7,

1971–1980

9 Thompson, and Blakesley, (1983) Purification of nucleic acids by RPC-5 analog

chromatography: peristaltic and gravity-flow applications Meth Enzymol 110,

123–127

10 Office of Vaccine Research and Review (1996) Points to consider on plasmidDNA vaccines for preventive infectious disease indications, Food and DrugAdministration, Bethesda, MD

11 Middaugh, C R., Evans, R K., Montgomery, D L., and Casimiro, D R (1998)

Analysis of plasmid DNA from a pharmaceutical perspective J Pharm Sci 8,

130–146

From: Methods in Molecular Medicine, vol 29, DNA Vaccines: Methods and Protocols

Edited by: D B Lowrie and R G Whalen Humana Press Inc., Totowa, NJ

of plasmid DNA (see Note 1) One main issue is that the process should

con-form to cGMP guidelines and be acceptable to the FDA or other national latory agencies The cGMP environment should be implemented independently

regu-of the intended use regu-of the DNA product

A typical application would be the supply of genetic information that is ing within the cell, e.g., because of a genetic disease like cystic fibrosis (CF)

miss-In such cases the “therapy” has been performed by transferring mid DNA complexes to the lung epithelium to express the absent chloride chan-

liposome-plas-nel gene (CFTR) for the restoration of the draining system of this tissue (1).

The more preventive approach of “gene medicine” could involve vaccination

using plasmid DNA either by subcutaneous or intramuscular injection (2–6) or other techniques (for an overview see ref 7, and this volume) The expression

of immunogenic epitopes can cause both humoral and CTL response (8,9), and

Chapter 6

In all cases, it is essential to be able to use a therapeutic agent (the logic,” usually a plasmid DNA) free of any other materials Such contamina-tion includes components used in the isolation process and coming from theorganism from which the plasmid is isolated, mainly residual proteins, RNA,and genomic DNA of the host cell In this chapter, we describe the develop-ment of a pharmaceutical manufacturing process to isolate plasmid DNA start-

“bio-ing from a technology (Qiagen, Hilden, Germany) that has been shown overthe past years to be a tool in research and development that fulfills the require-

ments described in Subheading 1 (see Notes 2–4).

1.1 The Host Cell Selection

A single appropriate host strain for all research work or industrial scale

phar-maceutical manufacturing does not exist (see Note 5) An appropriate strain

should be a clone derived from a host strain stock that is completely ized and free of any contamination It should be safe for the environment, forthe isolated product, the exposed patients, and the employees doing the manu-facturing work as well as health care personnel Considerable experience in thefield of molecular cloning and DNA techniques has been obtained with

character-Escherichia coli (E coli), and E coli K12 fulfills the needs for a safe,

well-characterized host strain for DNA production

Systematic analysis of over 20 different E coli substrains demonstrated that

very large qualitative and quantitative differences exist among all the substrainstested These differences mainly concern particularly the amount of plasmidDNA per gram of biomass and the plasmids isoform distribution

Plasmid isoforms consist of supercoiled molecules, dimers, or concatemers

or catenanes (chains of two or more plasmids), as well as linear or nicked mids The observed differences in isoforms depend on the plasmid as well as

plas-on the host strain This means that not plas-only the genetic background of the host

is responsible for the differences of the plasmid isoform distribution, but theplasmid itself also contributes to a certain extent

1.2 Growth Conditions

Bacterial cultures for the purpose of plasmid isolation were performed in abatch mode, using culture bottle volumes of up to a maximum of 2 L Studieswere done to determine the growth medium and conditions for optimal bacte-rial growth and plasmid yields, resulting in optical density (O.D 600) values

of around 3–6 O.D units in complex bacterial growth media For purposessuch as research grade plasmid preparations for cloning, sequencing, and trans-fection experiments, this procedure is adequate and the final analysis of theprepared DNA is usually an agarose gel electrophoresis, DNA quantificationand identity test (restriction digestion)

These test criteria are not stringent enough for pharmaceutical purposes,and the procedure of the manufacturing had to be drastically modified Thefirst point to consider in the development of a pharmaceutical grade process isthat the batch culture method has no type of online monitoring or regulation.The growth conditions are adjusted before inoculating the medium and leftunchanged, usually for between 16–20 h No pH monitoring or adjustment is

performed, oxygen and carbon source are also neither monitored nor regulated.Essential substrates are depleted and toxic products accumulate Degraded cel-lular components, including plasmids, accumulate in overgrown cultures andcell death follows.

To overcome these problems for the isolation of recombinant proteins,high performance fermentation technology has been developed over the lastfew years

Fermentation processes require different growth media than batch cultures.The possibility of monitoring the growth conditions allows for the introduction

of essential media components before they are exhausted (feeding), and for themaintenance of a constant pH control and oxygen supplies Besides the effects

of such regulation, the culture process becomes more defined and the ceutical requirements on documentation can be fulfilled A further feature ofthe fermentation technology for the large-scale plasmid production, is thepotential of high-density fermentation to yield large amounts of biomass.Experimental work on the composition of bacterial growth media for bottlecultures and fermentation demonstrates that the choice of fermentation condi-tions and growth media strongly influence the yields of plasmid that are

pharma-obtained from E coli cells One main focus was put on the amount of plasmid

per cell (copy number), that can be monitored on-line by capillary gel

electro-phoresis (10).

1.3 Downstream Processing

The isolation of a biomolecule from the bacterial culture (usually referred to

as downstream processing or DSP) is performed to separate plasmid DNA fromother undesired components present within the source of material These unde-sirable components are genomic DNA, RNA, proteins, lipids, lipopoly-saccharides (LPS) or endotoxins, components of the cell wall and intact

bacteria (see Note 6) The alkaline lysis (11) was modified and is reproducibly

performed in scales up to five liter bacterial culture (ultrapure 100 graphy system, Qiagen) The most important feature of this technique is theformation of a complex of most of the undesired components mentioned above,which can easily be removed by centrifugation (research scale) or floating andfiltration (research and industrial scale)

chromato-The resulting “cleared lysate” is applied to an industrial scale process matography column with anion exchange resin, to specifically bind the nega-tively charged plasmid DNA and (under appropriate buffer conditions) not tobind residual undesired components (e.g., protein, RNA, nucleotides, LPS).Such anion exchange chromatography is not limited in scale (compared toapproaches such as gel filtration) Moreover, in the case of specific types of resinmaterial, possessing dense, high surface charge, a one-step process can be used

chro-As an additional pharmaceutical requirement, a process for the complete, rapid

removal of LPS molecules was developed (see Subheading 2.5.) Endotoxins

such as E coli LPS can have cytotoxic effects on mammalian cells in vitro and in

vivo (12–15) and if present in large enough amounts in vivo can cause symptoms

of toxic shock syndrome and activation of the complement cascade (16).

In our process development we focused on the use of only non-toxic stances, and in particular avoided any potentially carcinogenic or immuno-genic reagents Additionally, the environment was controlled and the resultingliquid waste was biodegradable

sub-1.4 Quality Assurance and Quality Control

When we began our work on DNA manufacturing, the only criteria for thequality of plasmid DNA were those of typical research work Usually the qual-ity for this research grade material was estimated using analytical gel electro-phoresis, restriction enzyme digestion, and DNA sequence readings

We therefore established a set of quality criteria (17) that is now well

accepted by the scientific community Relevant issues from this work werediscussed at the Food and Drug Administration (FDA)/World Health Organi-zation (WHO) conference on Nucleic Acid Vaccines, February 5–7, 1996 atthe National Institute of Allergy and Infectious Diseases/National Institutes of

Health (NIAID/NIH) (Bethesda, MD) (18) An overview of the regulatory

as-pects for design, manufacturing, quality assurance, and quality control of cination vectors are summarized in the WHO “Guidelines for Assuring theQuality of DNA Vaccines” (WHO Technical Report, Jan 17, 1997)

vac-The design of the production process focused on its acceptance by nationaland international authorities such as the FDA (Washington, DC), MedicinesControl Agency (UK) and others, and had to fulfill the appropriate cGMP regu-

lations (see Note 7).

5 QC: 1.0 M NaCl, 50 mM MOPS, pH 7.0, 15% (v/v) isopropanol.

6 QN: 1.6 M NaCl, 50 mM MOPS, pH 7.0, 15% (v/v) isopropanol.

2.2 Transformation and Host Cells

Prepare competent cells such as E coli K12 DH5α (Life Technologies,Eggenstein, Germany), DH10B (Life Technologies) or TG1 (# 6056; Deutsche

Sammlung von Mikroorganismen und Zell Culturen, Braunschweig, many), transform the plasmid DNA, and select recipients on agar plates con-taining the appropriate selection factor.

Cells can be harvested by batch centrifugation at 4600g for 15 min at 4°C

1 Beckman J2-21 centrifuge with a JA-10E rotor

2 500 mL Polypropylene bottles (Nalgene, Rochester, NY)

2.5 The Anion Exchange Chromatography System

Perform anion exchange chromatography (Qiagen) to specifically bind doublestranded DNA Single stranded DNA, RNA, nucleotides, proteins, LPS and othercontaminants do not bind to the chromatographic resin under appropriate conditions

1 For small-scale preparations (e.g., test runs using the produced biomass), 500 µgbatches of DNA use the Qiagen EndoFree Plasmid Kit (Ref #12362)

2 For larger scale preparations, use an anion-exchange chromatography column forthe isolation of up to 100 mg plasmid DNA (e.g., ultrapure 100 column #11100,Qiagen) and LPS-free processing buffers (#11910, Qiagen)

3 Methods

The complete process of plasmid DNA production is performed under documented conditions and in the case of GMP manufacturing under controlledenvironmental conditions The following examples of the process we use will

well-give some insight into the steps performed (Fig 1).

3.1 The Host Cell Selection

To obtain a pure and well-characterized production strain capable of high

yields of DNA, the selection of an appropriate E coli K12 plasmid host cell

clone is essential Besides good microbiological practices and the use of standardoperating procedures (SOPs), a well established quality assurance and qualitycontrol system is of great relevance, since all further process steps depend on this

1 Check the DNA received by the Qiagen DNA Production Facility for large-scalemanufacturing for its identity first (size, restriction pattern, sequence), and if it issatisfactory, release it for further processing

2 Transform the DNA to E coli K12 host cells, and select individual colonies for

further cultivation

Fig 1 Flow chart of a cGMP plasmid manufacturing procedure “QA” cates the types of quality assurance tests that must be performed at certain steps toensure consistent quality and reproducibility, and to fulfill the needs of processdocumentation.

indi-3 Use 3 mL of an overnight culture of cells for a small-scale plasmid isolation(QIAprep, Qiagen) In case of large numbers of clones, use an automated devicefor the isolation of DNA in a 96-well format (BioRobot 9600, Qiagen)

4 Identify appropriate cell clones by comparing them and selecting those with highplasmid yield and a proper plasmid isoform distribution for further production

steps (Fig 2).

5 Further purify the selected clone by two single-colony passages and check it foridentity and absence of microbiological contaminants Use it subsequently forthe inoculation of a culture to prepare a glycerol stock of between 100–500 vials.This stock is called Master Cell Bank (MCB); it is necessary to be able to repro-ducibly inoculate culture media from the MCB in the following process step andany future manufacturing run.

6 Perform an extensive quality assurance program to check the quality of this MCB.Test the identity, plasmid content, as well as absence of microbiological con-taminants before proceeding with the following step An important additionalrequirement is the complete sequencing of the DNA construct at this stage toexclude any difference to the original plasmid and to have a data backup for post-production sequencing

7 Use vials of the MCB to inoculate a fresh culture to produce an equally large set

of stocks (100–500 vials), which are required for the reproducible inoculation ofthe fermentation-precultures This second glycerol stock is called ManufacturingWorking Cell Bank (MWCB) Perform the same tests for Quality Assurance (QA)

as with the MCB

3.2 Fermentation

A fermentation process for E coli cells carrying plasmids in a certain copy

number must be well characterized, reproducible, easy to monitor and late; If possible it should run automatically The MCB and MWCB describedabove are the backbones for any reproducibility Further important issues are

regu-Fig 2: Comparison of plasmid pUC21 DNA produced in different E coli K12 host

cells The upper panel shows the undigested DNA with its different isoforms The

lower panel shows as a control the same amount of DNA after an EcoRI digestion The molecular size marker (M) is HindIII digested λ-DNA Gel: 1% (w/v) agarose in TAE,

pH 8.0, run at 5V/cm and stained with ethidium bromide

the types of fermenter, regulation and growth medium used Batches of 5–200

L are routinely run, and if required, further scaling-up is possible

1 Use an appropriate amount of the MWCB to inoculate a pre-culture in E coli

To isolate the plasmid DNA from the E coli cells, a modified alkaline lysis

procedure (11) is used This step is of critical importance to reduce

contami-nants such as protein, RNA, genomic DNA, and cell wall residues Here wedescribe, as a pilot scale example, the approach of isolating up to 100 mg plas-mid DNA starting from 60 g wet weight biomass (see also the protocol sup-plied with the Qiagen ultrapure100 kit)

1 Thoroughly resuspend 60 g biomass in 1000 mL buffer P1 in a 5-L glass bottle

2 Add 1000 mL of buffer P2, mix the complete volume and incubate it at roomtemperature for 5 min

3 Add 1000 mL of buffer P3 and mix it carefully

4 Incubate the lysate for 30 min at room temperature to allow the flaky white cipitate of SDS, protein, genomic DNA, and cell residue to rise to the surface

pre-5 Carefully pump the lysate out of the bottle

6 Filter the lysate through a QIAfilter™ unit (Qiagen), mixed with 1/10 volume ofbuffer ER (Qiagen), and collect the filtrate for subsequent chromatography

3.4 Anion Exchange Chromatography

The anion exchange chromatography columns are loaded by pumping lysatewith a peristaltic pump or preferably with a process chromatography systemfor better monitoring of the process

1 Equilibrate the Qiagen ultrapure 100 column with 350 mL buffer QBT at a flowrate of 10 mL/min

2 Load the column at a flow rate of approximately 4 mL/min overnight

3 Wash the charged column with 3 L of LPS-free buffer QC at a flow rate of

20 mL/min

4 Elute plasmid DNA with 400 mL LPS-free buffer QN at a flow rate of 3 mL/min

5 Precipitate the DNA with 0.7 volumes of isopropanol at 4°C and centrifuge at

20,000g for 30 min in LPS-free centrifuge bottles.

6 Wash the DNA pellet with LPS-free 70% EtOH and rinse

7 Dry the DNA pellet and resuspend it in the appropriate buffer system for furtherapplications

differ-of the sequence.

2 Sequencing: Determine the complete nucleotide sequence of both DNA strands

by DNA sequencing Perform all steps following SOPs and document the data in

a sequencing report

3 Plasmid Stability: Monitor the presence or absence of a plasmid containing anantibiotic resistance marker by inoculating a defined amount of cells on bothantibiotic and non-selective agar plates If cells are not able to grow on selectiveplates, the percentage of clones growing on both media represents the “plasmidstability.”

4 DNA Quality: In addition to the analyses of fragment identity and sequence, usespectrophotometric scans between 220–320 nm for the detection of salt and

organic contamination (20) within the DNA Inspect the appearance of a sample

in an agarose gel electrophoresis Important features are the isoform distribution(by agarose gel electrophoresis) and the DNA concentration Also determine thecontent of RNA, genomic DNA and LPS by HPLC, Southern blot and the kineticQCL test kit (BioWhittaker, Walkersville, MD) respectively

5 DNA Quantity: Determine the DNA concentration by spectrophotometric sis and calculation from its absorbance at 260 nm

analy-4 Notes

1 For large scale DNA production, we focused on the development of a technologyfor industrial-scale manufacturing of nucleic acids that combines cost effective-ness with the flexibility to install the system in every research laboratory (pilotscale) or GMP facility (industrial scale)

2 A major consideration in the development of this technology was to avoid consuming centrifugation and multiple chromatographic column runs Centrifu-gation of large volumes to clear bacterial lysates can now be replaced by just onepassage through a filtration unit that makes it possible to filter large volumes ofbacterial lysate

time-3 The process includes the establishment of Master Cell Banks and Master ing Cell Banks; fermentation and downstream processing are monitored at allstages by extensive in-process controls

Work-4 The three most important factors which need to be considered in the processdevelopment for plasmid DNA production are: selection of the optimal host strain,optimization of growth conditions, and the nucleic acid preparation method

5 A large set of different E coli host strains has been studied to identify strains

producing large amounts of plasmid DNA per cell with the highest quality ity criteria for the selection of a host strain are the homogeneity of the plasmidDNA isolated from the host strain (>90% covalently closed circle), and theendotoxin content of the DNA purified from a specific host strain

Qual-6 Endotoxins (LPS) are major contaminants of nucleic acids, especially plasmidDNA preparations Due to their negatively charged phosphate groups, endotoxinstend to co-purify with nucleic acids It has been demonstrated that LPS contami-nation of DNA has a direct influence on transfection efficiency into many types

of cultured cells, and that different cells show variable sensitivity to this

con-tamination (13).

7 The Qiagen procedure has been approved to produce DNA for human clinical

Phase I studies in the UK (1) and other European countries, as well as in the United States by the FDA (21) A drug master file (DMF) for the clinical grade

manufacturing process is filed with the FDA

2 Davis, H L., Whalen, R G., and Demeneix, B A (1993) Direct gene transfer

into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of

expression Hum Gene Ther 4, 151–159.

3 Manthorpe, M., Cornefer-Jensen, F., Hartikka, J., Felgner, J., Rundell, A.,Margalith, M., and Dwarki, V (1993) Gene therapy by intramuscular injection of

plasmid DNA: studies on firefly luciferase gene expression in mice Hum Gene

Ther 4, 411–418.

4 Michel, M.-L., Davis, H L., Schleef, M., Mancini, M., Tiollais, P., and Whalen,

R G (1995) DNA-mediated immunization to the hepatitis B surface antigen inmice: aspects of the humoral response mimic hepatitis B viral infection in humans

Proc Natl Acad Sci USA 92, 5307–5311.

5 Davis, H L., Michel, M.-L., Mancini, M., Schleef, M., and Whalen, R G (1994)Direct gene transfer in skeletal muscle: plasmid DNA-based immunization against

the hepatitis B surface antigen Vaccine 12, 1503–1509.

6 Wolff, J A., Williams, P., Acsadi, G., Jiao, S., Jani, A., and Chong, W (1991)Conditions affecting direct gene transfer into rodent muscle in vivo

BioTechniques 11, 474–485.

7 Wolff, A J (1994) Gene Therapeutics—Methods and Applications of Direct Gene

Transfer, Birkhäuser, Boston.

8 Schirmbeck, R., Böhm, W., Ando, K., Chisari, F V., and Reimann, J (1995)Nucleic acid vaccination primes hepatitis B surface antigen-specific cytotoxic T

lymphocytes in nonresponder mice J Virol 69, 5929–5934.

9 Davis, H L., Schirmbeck, R., Reimann, J., and Whalen, R.G (1995) ated immunization in mice induces a potent MHC class I-restricted cytotoxic T

DNA-medi-lymphocyte response to hepatitis B virus surface antigen Hum Gene Ther 6,

1447–1456

10 Schmidt, T., Friehs, K., and Flaschel, E (1996) Rapid determination of plasmid

copy number J Biotech 49, 219–229.

11 Ish-Horowics, D., and Burke, J F (1981) Rapid and efficient cosmid cloning

Nucleic Acid Res 9, 2989–2998.

12 Cotten, M., Baker, A., Saltik, M., Wagner, E., and Buschle, M (1994) charide is a frequent contamination of plasmid DNA preparations and can be toxic

Lipopolysac-to primary cells in the presence of adenovirus Gene Ther 1, 239–246.

13 Weber, M., Möller, K., Welzeck, M., and Schorr, J (1995) Effects of

lipopolysac-charide on transfection efficiency in eukaryotic cells BioTechniques 19, 930–940.

14 Wicks, I P., Howell, M L., Hancock, T., Kohsaka, H., Olee, T., and Carson, D

A (1995) Bacterial lipopolysaccharide copurifies with plasmid DNA:

implica-tions for animal models and human gene therapy Hum Gene Ther 6, 317–323.

15 Morrison, D C and Ryan, J L (1987) Endotoxins and disease mechanisms Annu.

17 Schorr, J., Moritz, P., Seddon, T., and Schleef, M (1995) Plasmid DNA for

hu-man gene therapy and DNA vaccines NY Acad Sci 772, 271–273.

18 Smith, H A., Goldenthal, K L., Vogel, F R., Rabinovich, R., and Aguado, T.(1997) Workshop on the control and standardization of nucleic acid vaccines

Vaccine 15, 931–933.

19 Miller, J H (1972) Experiments in Molecular Genetics, Cold Spring Harbor

Labo-ratory Press, Cold Spring Harbor, NY, p 443

20 Wilfinger, W.W., Mackey, K., and Chomczynski, P (1997) Effect of pH and ionicstrength on the spectrophotometric assessment of nucleic acid purification

From: Methods in Molecular Medicine, vol 29, DNA Vaccines: Methods and Protocols

Edited by: D B Lowrie and R G Whalen Humana Press Inc., Totowa, NJ

3

Development and Characterization

of Lyophilized DNA Vaccine Formulations

Nancy L Shen, Jukka Hartikka, Nancy A Horn,

Marston Manthorpe, and Magda Marquet

1 Introduction

The potential applications of using plasmid DNA for immunization and othergene therapy approaches have been discussed in an increasing number of pub-lications in the past few years Injection of mouse muscle with naked DNA(plasmid DNA in saline) resulted in significant episomal expression from anumber of encoded reporter genes such as firefly luciferase, chloramphenicolacetyltransferase, and β-galactosidase (1) DNA vaccination has been shown

to induce neutralizing antibodies against the gene product, helper T-cell

responses of the Th1 phenotype, and cytotoxic T lymphocyte responses (2).

Vaccination with plasmid DNA stimulates immunogenicity and provides tection against various infectious diseases in pre-clinical animal models

pro-Examples include hepatitis B in chimpanzees (3), bovine herpes virus in mice (4), influenza A virus in ferrets (5), human immunodeficiency virus in rhesus monkeys (6), Mycobacterium tuberculosis in mice (7,8), malaria in mice (9,10), and genital herpes simplex virus in guinea pigs (11) Recently, DNA vaccines

for the protection against influenza (Merck Research Laboratories, Rahway,NJ), malaria (Vical Inc., San Diego, CA), and HIV (Apollon Inc., Philadel-phia, PA), have entered phase I human clinical trials Rapid progress has been

made in the areas of adjuvants for DNA vaccines (12), route of immunization (13), industrial scale fermentation and pharmaceutical grade purification (14).

One major interest in the commercial development of DNA vaccines, cially for developing countries, is to increase DNA vaccine stability at room

espe-temperature, to reduce the requirement for costly cold storage, and to extendproduct shelf-life.

Freeze-drying, or lyophilization, has been used in pharmaceutical processes

to prolong product stability, particularly for protein products (15,16) drying is used for an attenuated virus vaccine against yellow fever (17) and a live rinderpest virus vaccine for cattle (18) The freeze-drying process can be

Freeze-divided into three successive stages: freezing, primary drying, and secondarydrying After freezing the product, the primary drying process involves lower-ing pressure and supplying heat for water vapor sublimation During the sec-ondary drying stage, the residual absorbed moisture evaporates from the driedmaterial In this chapter, we describe a lyophilized DNA vaccine formulationthat provides acute protection during lyophilization and permits a full recovery

of product activity

1.1 Screening Buffer and pH

To screen excipients used in lyophilized vaccine DNA formulations, weevaluated buffers and pH using an in vivo reporter gene assay Plasmid DNA

VR1223 encoding a gene for luciferase (19,20) was formulated and injected

intramuscularly into adult mouse rectus femoris muscle at 50 µg DNA in

50 µL volume Injections were performed on 5 mice (10 muscles) for eachformulation Luciferase enzyme activity was measured 7 d post-injection The

results are shown in Table 1 There was no statistical difference in expression

from any of the tested formulations by non-parametric Mann-Whitney rank

sum test (p < 0.05) Either pH 6.0 or pH 7.0 was appropriate Similarly, either

phosphate-buffered saline (PBS) or citrate-buffered saline permitted geneexpression There were no adverse effects detected in injected mice

1.2 Screening Lyoprotectants

Lyoprotectant, a required component in a lyophilized formulation, providesprotection of biological molecules from freezing and drying processes andgives mechanical support to the finished product To screen and selectlyoprotectants, various sugars and polymers were added to the liquid formula-

tion and tested in mouse muscles The results are summarized in Table 2.

Considerable variation in luciferase enzyme expression was noted among ous concentrations of sugars or polymers, and among similar experiments per-formed by using different batches of mice There was no statistical difference inexpression from any of the tested lyoprotectants compared to PBS, pH 7.0, by

vari-non-parametric Mann-Whitney rank sum test (p < 0.05) However, a 2- to 3-fold

enhancement was observed from formulations containing sugar lyoprotectant orsugar/polymer lyoprotectant The sugars included trehalose, mannitol, lactose,sucrose, and sorbitol; and the polymers included polyvinyl pyrrolidone (PVP)

K-30 and polyethylene glycol (PEG)-3350 No adverse effect on these micewas observed during the 7 d following injection of the test formulations.Trehalose, PVP K-30, and PEG-3350 were chosen for further vaccine for-mulation development Trehalose is a non-reducing disaccharide of glucose

found in several organisms that are able to survive desiccation (21) Trehalose

has been demonstrated to protect cells from freezing injury and is an effective

drying protectant (22) Commercial formulations containing trehalose and PVP

have shown good recovery of enzyme activity after freeze-drying and morethan one year storage stability at 2–8°C (23).

1.3 Freeze-Drying Cycle Development

Next, we characterized two proposed formulations by freeze-drying

micros-copy (24) and examined the effect of lyophilization parameters on the finished

freeze-dried product to determine a freeze-drying cycle Sodium phosphate

crystallizes during freezing and causes pH shifts (25) Sodium chloride has a

rather low eutectic temperature and requires a rather low primary drying

tem-perature (25) Therefore, sodium phosphate and sodium chloride were removed

from the proposed lyophilized formulation, but both components were includedlater in the reconstituting reagent Freeze-drying microscopic analysis was per-formed on two formulations, and the results indicated that the retention of cake-like structure in the dried region was observed below the collapse temperature.The collapse temperature for formulation A (VR1223 DNA [1 mg/mL]/12%trehalose [w/v]/2% PVP [w/v]) was –26°C The collapse temperature for for-mulation B (VR 1223 DNA [1 mg/mL]/15% trehalose [w/v]/2% PVP [w/v]/2% PEG [w/v]) was –27°C A complete loss of structure was observed abovethe collapse temperature (Dr Michael Pikal, University of Connecticut, Storrs,

Table 1

Screening Buffers and pH Used in DNA Vaccine Formulations

Average± SEM:

ng of luciferase per Total number

0.9% NaCl, 10mM sodium phosphate, pH 7.0 228.2 ± 76.9 10

0.9% NaCl, 10 mM sodium citrate, pH 7.0 216.9 ± 37.6 10

0.45% NaCl, 10mM sodium citrate, pH 7.0 178.6 ± 67.9 10

0.45% NaCl, 10 mM sodium citrate, pH 6.0 137.9 ± 35.7 10

All formulations contained VR1223 plasmid DNA at 1 mg/mL concentration Luciferase was extracted from mouse leg muscle and assayed 7 d post-injection None of these values were signifi-

cantly different from saline control by non-parametric Mann-Whitney rank sum test (p < 0.05).

CT, personal communication) Therefore, the primary drying temperature wasmaintained below the collapse temperature by adjusting the chamber pressure

to 53 µm Hg As the sublimation of ice was completed, the product ture was increased to +35°C for 12 h during the secondary drying This processresulted in drying of 1 mL of liquid DNA formulation within 2 d

tempera-1.4 Physical Chemical Characterization

A summary of results from tests on the finished product is shown on Table 3.

The lyophilized product is a white “cake” and partially detached from the glassvial The cake contains approximately 2% water by weight The lyophilizedcake can be reconstituted with 1 mL of PBS, pH 7.0 ± 0.2 The lyophilized

Table 2

Screening Lyoprotectants

by Using In Vivo Mouse Muscle Luciferase Assay

Average±SEM: ng of Total Percent ofluciferase number of PBS

PBS/12% lactose/0.9% benzoyl alcohol 128.1 ± 40.5 10 89All formulations contained VR1223 plasmid DNA at a concentration of 1 mg/mL The pH of all formulations was 7.0

aTotal number of muscles was from 1–5 experiments None of the results were significantly

different from PBS control by non-parametric Mann-Whitney rank sum test (p < 0.05).

cake dissolves within 1 min and forms a clear, colorless, and odorless solution.There was no detectable loss of DNA determined by spectrophotometricabsorption at 260 nm wavelength Supercoiled plasmid DNA structure wasretained without apparent degradation, as revealed by agarose gel electrophore-sis with ethidium bromide staining.

1.5 In Vitro Bioassays

of the Reconstituted Lyophilized DNA Formulation

Two plasmids, VCL1005 (26) and VCL1102 (27), were lyophilized and

tested for in vitro potency Both plasmids are currently being used in human

anti-cancer clinical trials (26,27) The VCL1005 encoding human HLA-B7

gene was lyophilized in formulation A (12% trehalose/2% PVP), and tuted with 1 mL PBS, pH 7.0 ± 0.2 The reconstituted VCL1005 formulationwas complexed with DMRIE/DOPE (1,2-dimyristyl-oxypropyl-3-dimethyl-hydroxyethyl ammonium bromide/dioleoylphosphatidylethanolamine) and

reconsti-used to transfect UM449 human melanoma tumor cells (28) Table 4 indicates

that the HLA-B7 is synthesized and expressed on the cell surface of UM449 at

48 h after transfection The level of HLA-B7 expression was not significantlydifferent from the expression of the non-lyophilized DNA control

Plasmid VCL1102 encoding the gene for human IL-2 was also lyophilized

in formulation A in addition to formulation B (15% trehalose/2% PVP/2%PEG) Freeze-dried vials were reconstituted with PBS, pH 7.0 ± 0.2 andcomplexed with DMRIE/DOPE, and both formulations were used to transfectUM449 cells The culture supernatant was assayed for human IL-2 by ELISA

(27) Table 5 indicates that IL-2 is synthesized and secreted in cell culture

Table 3

Physical-Chemical Test on Lyophilized and Reconstituted Vials

General appearance White cake, partially detached from

VCL1102 plasmid DNA was lyophilized in formulation A (12% trehalose, 2% PVP) or mulation B (15% trehalose, 2% PVP, 2% PEG) The freeze-dried vial was reconstituted with 1 mL PBS, pH 7.0.

for-supernatants 48 h post-transfection There is no significant difference in IL-2expression between lyophilized VCL1102 and non-lyophilized control DNA.

1.6 In Vivo Bioassays

of the Reconstituted Lyophilized DNA Formulation

A panel of lyophilized VR1223 plasmid DNA formulations were evaluatedfor luciferase expression in mice Lyophilized formulations contained treha-lose, PVP, and PEG lyoprotectants Freeze-dried vials were reconstituted withPBS, pH 7.0 ± 0.2 and injected into mouse rectus femoris muscles Luciferase

activities were assayed at 7 d after injection Results, as shown in Table 6,

indicate that the level of muscle expression from reconstituted formulation A(12% trehalose/2% PVP) is similar to non-lyophilized DNA control In addi-tion, there is a statistical difference in expression between formulation A andformulation F (8% trehalose/2% PVP/2% PEG) by non-parametric Mann-

Whitney rank sum test (p < 0.05) The reconstituted formulation A yields

3-fold higher expression than reconstituted formulation F There was nodetectable adverse side effect on mice for 7 d after injection

Table 4

Plasmid VCL1005 Lyophilized in Formulation A

Corrected mean Number of Percent

Control, non-lyophilized VCL1005 32.71 0.16 2 100Lyophilized VCL1005, reconstituted 34.65 4.17 2 106

VCL1005 was lyophilized in formulation A (12% trehalose, 2% PVP) and reconstituted in 1 mL PBS, pH 7.0 HLA-B7 expression was measured by FACS analysis by using an in vitro cell transfec- tion assay.

Table 5

Plasmid VCL1102 Lyophilized in Fomulation A or B

Average corrected Number Percent of

Lyophilized VCL1102 in formulation A 2508 710 4 128Lyophilized VCL1102 in formulation B 1720 297 4 88

Lyophilized VCL 1102 was reconstituted in 1 mL PBS, pH 7.0 Human IL-2 expression in the natant of UM449 cells after transfection was measured by ELISA Formulation A is: 12% trehalose, 2% PVP Formulation B is: 15% trehalose, 2% PVP, 2% PEG.

super-1.7 Conclusion and Perspectives of Lyophilized DNA Vaccines

The experimental work presented in this chapter describes development of alyophilized naked DNA vaccine for potential use in human gene therapy.Freeze-dried live or attenuated viral vaccines for yellow fever and rinderpest

diseases in cattle were previously reported (17,18) In this report, we evaluated

various sugars as lyoprotectants, particularly the non-reducing disaccharides,such as trehalose, and water soluble polymers, such as PVP and PEG A freeze-drying cycle for plasmid DNA formulation was developed based on freeze-drying microscopic analysis and an examination of the effect of lyophilizationparameters Typical finished cake was white and readily dissolved in PBS,

pH 7.0 Recovery of full product activity was demonstrated by plasmid DNAgene expression from mammalian cell culture in vitro and from mouse muscle

in vivo Thus, the freeze-drying process did not introduce any adverse effect

on the integrity and potency of plasmid DNA

The results suggest that a lyophilized DNA vaccine formulation is cially feasible Stability, sterility, and toxicity studies are currently being pur-sued and are required prior to clinical development The procedures describedherein may be used to prepare a safe and effective lyophilized DNA vaccinewith a shelf-life that is appropriate for clinical use

commer-Table 6

Plasmid VR1223 Lyophilized Formulations

Average± SEM: Total

ng of luciferase number of

Control, non-lyophilized VR1223 in PBS, pH 7.0 202.9 ± 54.1 10

Lyophilized VR1223 in formulation Aa 265.9± 60.4 10Lyophilized VR1223 in formulation B 184.5 ± 63.5 10Lyophilized VR1223 in formulation C 172.7 ± 56.2 10Lyophilized VR1223 in formulation D 136.0 ± 50.6 10Lyophilized VR1223 in formulation E 131.4 ± 61.4 10

Lyophilized VR1223 in formulation Fa 98.4± 41.9 10Lyophilized VR1223 in formulation G 90.7 ± 21.1 10Lyophilized formulations were reconstituted with 1 mL PBS, pH 7.0 and injected in mouse leg muscle Luciferase activity was assayed 7 d after injection Formulation A is: 12% trehalose, 2% PVP Formulation B is: 15% trehalose, 2% PVP, 2% PEG Formulation C is: 10% trehalose, 2% PVP, 2% PEG Formulation D is: 12% trehalose, 2% PVP, 2% PEG Formulation E is: 15% trehalose, 2% PVP Formulation F is: 8% trehalose, 2% PVP, 2% PEG Formulation G is: 10% trehalose, 2% PVP.

aThe result with formulation A is significantly different from formulation F by ric Mann-Whitney rank sum test (p < 0.05).

non-paramet-2 Materials

2.1 Chemicals

All ingredients in these formulations are approved drug substances for injection

1 Trehalose, mannitol, lactose, PVP K-30, PEG-3350, sorbitol, and sucrose areUSP grade and from Spectrum Quality Products (New Brunswick, NJ)

2 USP grade sterile water for injection (SWFI) and sodium chloride (5%) in SWFIare from Baxter Healthcare (Round Lake, IL)

3 Sugar and buffer stock at concentrations of 20–40% are prepared in SWFI andfiltered through 0.22 µm filters (Nalgene, Rochester, NY)

4 Plasmid DNA is replicated and isolated from Escherichia coli DH10B strain (29) Supercoiled plasmid is purified by column chromatography (30) Plasmid con- structs are precipitated by ethanol (29), re-solubilized in SWFI, and stored at

–20°C Plasmid endotoxin levels are <30 endotoxin units/mL based on LAL gelclot assay (Associates of Cape Cod, Woods Hole, MA) and the spectrophotometric

A260/A280 ratios are between 1.8 and 2.0

2.2 Containers and Equipment

1 The 5-mL glass vials (Type 1 glass), 13-mm gray butyl stoppers, aluminum crimpseals, and a crimper are obtained from West Co (Lionville, PA)

2 A pilot freeze-dryer (Duro-stop MP) is available from FTS Kinetics (Stone Ridge,NY)

3 Karl Fisher water titration Aquastar apparatus is available from EM Science(Gibbstown, NJ)

4 A microplate luminometer, Dynatech Model ML250 is available from AnalyticalLuminescence Labs (San Diego, CA)

5 Statistical software such as SigmaStat version 2.0 is available from Jandel tific Software (San Rafael, CA)

Scien-6 FACS can/LYSIS II system is available from Becton Dickinson (Mountain View, CA)

2.3 Formulations and Filling Vials

Prepare formulations in a biological safety hood by mixing concentratedsterile stock solutions including desired plasmid DNA constructs, sugar, poly-mer, buffer, and 5% sodium chloride

1 Prepare 0.2 M sodium phosphate by dissolving 3.86 g of sodium phosphate

diba-sic and 0.77 g of sodium phosphate monobadiba-sic in 100 g of SWFI Prepare PBS,

pH 7.0 ± 0.2 by mixing SWFI, 0.2 M sodium phosphate at pH 7.0 ± 0.2, and 5%

sodium chloride (in SWFI) to achieve final concentrations of sodium phosphate at

10 mM and sodium chloride at 0.9% Filter stock solutions through a 0.22 µm filter

2 Wash and rinse glass vials and stoppers with SWFI Bake glass vials at 225°C in

a depyrogenation oven for at least 4 h Autoclave glass vials and stoppers beforeformulation filling

3 Dispense formulations in aliquots of 1 mL into 5-mL glass vials (Type 1 glass,West Co.)

2.4 Plasmid Constructs

1 Plasmid DNA VCL1005 (26) contains the Rous Sarcoma virus long terminal

repeat (RSV-LTR) promoter/enhancer that drives the transcription and tion of the MHC class I human leukocyte antigen B7 (HLA-B7) gene and thechimpanzeeβ2 microglobulin gene in a pUC19 backbone Plasmid VCL1005 alsopossesses polyadenylation sequences from bovine growth hormone and kanamy-cin resistance gene as a bacterial selection marker

transla-2 Plasmid VCL1102 (27) contains the coding sequence for the human

interleukin-2 (IL-interleukin-2) gene that is downstream from the CMV immediate-early gene promoter/enhancer and 5' untranslated sequences in a pUC18 backbone

3 Plasmid VR1223 (19,20) encodes the Photinus pyralis luciferase gene under the

control of human CMV immediate-early promoter with intron A in a partiallydeleted pBR322 VR1223 contains a kanamycin-resistance sequence as a bacte-rial selection marker

3 Methods

3.1 Freeze-Drying Methods

1 Determine the collapse temperature of the formulation by using freeze-drying

microscopic analysis (24) (see Note 1).

2 Place the vials on the freeze-drier shelves at room temperature and subsequentlyequilibrate at –1°C for about 30 min

3 Cool the shelves to –55°C and hold that temperature for 2 h

4 Carry out primary drying at a product temperature of about –32°C, or 5°C belowthe collapse temperature

5 Carry out secondary drying at 35°C Complete the drying after adjusting thechamber pressure to between 55–120 µm Hg

6 Insert the stoppers into the vials under vacuum in the freeze-dryer Crimp-sealthe freeze-dried vials and store them at 2–8°C

3.3 Physical-Chemical Characterization

1 Visually inspect the lyophilized “cake” for the color, texture, and volume under atranslucent light against a black background

2 Analyze the moisture content using Karl Fisher water titration

3 Reconstitute the freeze-dried cake with 1 mL of PBS, pH 7.0 ± 0.2 and visuallyinspect the solubility

4 Estimate plasmid DNA recovery using agarose gel (0.8%) electrophoresis (29)

alongside a control of non-lyophilized plasmid DNA standard for comparison.Determine plasmid DNA concentration and recovery by spectrophotometric

absorption at 260 nm (29).

3.4 In Vivo Bioassays

1 Inject formulated plasmid VR1223 (50 µg) intramuscularly in the rectus femoris

muscle of female 4- to 12-wk-old BALB/c mice (Harlan Sprague-Dawley) (see

Note 2).

2 At 7 d post-injection, extract the luciferase from the entire quadriceps muscle and

assay using an automated microplate luminometer (20).

3 Perform statistical comparisons using the non-parametric Mann-Whitney ranksum test

3.5 In Vitro Bioassays

1 Reconstitute freeze-dried vials containing plasmid DNA VCL1005 with 1 mLPBS, pH 7.0 ± 0.2

2 Complex the plasmid DNA (5 µg) with DMRIE/DOPE in 1:1 lipid mixture to yield

a DNA/lipid mass ratio of 1:1 to enhance introduction of the DNA into cells (28).

3 Use the DNA/lipid mixture to transfect UM-449 human melanoma cells (see

Note 3).

4 Harvest transfected cells after 48 h of incubation and subject them to fluorescent staining with HLA-B7 monoclonal antibody (hybridoma BB7.1,

immuno-ATCC # HB56) and flow cytometric analysis (26).

5 Reconstitute freeze-dried VCL1102 with 1 mL PBS, pH 7.0 ± 0.2 to a final DNAconcentration of 1 mg/mL

6 Complex the plasmid DNA (30 µg) with 6 µg of DMRIE (as DMRIE/DOPE 1:1lipid mixture) to yield a DNA/lipid mass ratio of 5:1 Dilute the DNA/lipid mix-ture to 2 µg DNA/mL and transfect IL-2 deficient UM449 cells (28).

7 Harvest the culture supernatant after 48 h and test in triplicate for the presence ofIL-2 using an Enzyme Amplified Sensitivity Immunoassay kit (Medgenix Diag-

nostics, Fleurus, Belgium) (27).

3 UM-449 human melanoma cells do not express HLA-B7 molecules and aredeficient in expressing β2-microglobulin They were obtained as a gift fromMark Cameron, Alfred Chang, and Gary Nabel, University of Michigan, AnnArbor, MI

Acknowledgments

The authors would like to thank Research Production for providing DNA,Quality Control for analytical support, Francine Cornefert-Jensen and PamelaStrauch for technical support, and George Gray for continued guidance andsupport

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28 Felgner, J H., Kumar, R., Sridhar, C N., Wheeler, C J., Tsai, Y.J., Border, R.,Ramsey, P., Martin, M., and Felgner, P L (1994) Enhanced gene delivery and

mechanism studies with a novel series of cationic lipid formulations J Biol.

Chem 269, 2550–2561.

29 Sambrook, J., Fritsch, E F., and Maniatis, T (eds.) (1989) Molecular Cloning:

A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, NY

30 Horn, N A., Meek, J A., Budahazi, G., and Marquet, M (1995) Cancer gene

therapy using plasmid DNA: purification of DNA for human clinical trials Hum.

Gene Ther 6, 565–573.

From: Methods in Molecular Medicine, vol 29, DNA Vaccines: Methods and Protocols

Edited by: D B Lowrie and R G Whalen Humana Press Inc., Totowa, NJ

plasmid DNA from Escherichia coli We have found that by repeatedly

regen-erating the column and reapplying the flowthrough volume of DNA extract,

we can readily obtain 2–4-fold increased yields, up to 40 mg, from the standard2.5 L broth culture

Labo-3 LB agar plates: these are prepared as above but containing 15 g/L Bacto-agar anddispensed into sterile Petri dishes after addition of the antibiotic

3 Methods

1 Streak out the transfected bacteria on two LB agar plates, one containing therecommended concentration of antibiotic and one containing double the concen-tration Incubate the plates for 24 h at 37°C

2 Select five or six typical colonies growing at the highest concentration of otic and inoculate them separately into 10 ml LB broth and incubate the culturesfor 8–10 h.

antibi-3 Make minipreps of plasmid DNA from these samples and select the culture ing the highest yield

show-4 Inoculate 1.0 or 0.5 L volumes of LB broth (total 2.5 L) with 1.0 or 0.5 mL ofthe selected culture and incubate the cultures on an orbital flask shaker over-night at 37°C

5 Prepare the bacterial lysate and pass it through the Qiagen column as described inthe kit but retaining the flowthrough lysate

6 After eluting the plasmid from the column according to the protocol, wash thecolumn with 100 mL sterile distilled water and then with a 50 mL equilibration

buffer The column is now ready for reuse (see Note 1).

7 Rerun the flow-through lysate through the column, elute the retained plasmid,wash and reequilibrate the column as before

8 Repeat this step a further three times (see Note 2) Typical yields are shown

in Table 1.

4 Notes

1 The columns can be sealed with parafilm and stored at 4°C

2 The column can be used for a further 2.5 L of culture (of the same plasmid) withsomewhat reduced yield before the flow becomes too restricted

Reference

1 Tascon, R E., Colston, M J., Ragno, S., Stavropoulos, E., Gregory, D., and

Lowrie, D B (1996) Vaccination against tuberculosis by DNA injection Nat.

Med 2, 888–892.

Table 1

Sequential Yields of Plasmid DNA

Pass l Pass 2 Pass 3 Pass 4 Pass 5 Total yield

LPS contentb <0.05 <0.05 <0.005 <0.005 <0.0005

Immuno- NT 1.6 ± 0.3 2.4 ± 0.2 3.5 ± 0.5 NT

genicityc

a pCMV4.65 (1) was recovered from E coli lysate by passing the lysate through a Qiagen

tip-10,000 column five times (Qiagen).

bEndotoxin units/microgram assayed by limulus endotoxin assay kit.

c Balb/c mice were injected intramuscularly with 2 × 50 µg DNA four times at 4-wk intervals, then killed after 2 wk Splenocytes from two to three mice were pooled, cultured for 48 h with or without recombinant antigen (10 µg/mL), then supernatants were assayed for interferon-γ by

enzyme-linked immunosorbent assay (1) Results are shown as ng/mL ± SD increment in the presence of antigen.

From: Methods in Molecular Medicine, vol 29, DNA Vaccines: Methods and Protocols

Edited by: D B Lowrie and R G Whalen Humana Press Inc., Totowa, NJ

5

The Immunology of DNA Vaccines

Thomas Tüting, Jonathan Austyn, Walter J Storkus,

and Louis D Falo Jr.

1 Introduction

The surprising observation that direct inoculation of an expression plasmidencoding a foreign protein into the skin of mice resulted in the induction ofantibody responses, demonstrated that injection of “naked” DNA could result

in antigen expression in an immunogenic form (1) This observation and the

subsequent demonstration that intramuscular injections of plasmid DNAencoding influenza nucleoprotein could protect mice against a challenge with

live influenza virus have opened up new avenues for vaccine development (2–3).

Immunization with plasmid DNA has been shown to activate both humoral andcellular immune responses, including the generation of antigen-specific CD8+cytotoxic T cells as well as CD4+ T helper cells (4) An increasing number of

studies using experimental animal models have demonstrated that plasmidDNA immunization can promote effective immune responses against numer-ous viruses, including influenza, rabies, HIV, HBV, HCV, and HSV; several

bacteria, including: Mycobacterium tuberculosis, Mycoplasma pulmonis, and

Borrelia burgdorferi; as well as parasites, such as malaria and leishmania (4).

Phase I clinical vaccine trials are currently being performed for HIV, HBV,

and influenza virus With the molecular identification of tumor antigens (5),

there has also been increasing interest in the development of DNA-basedimmunization for cancer Preclinical studies demonstrate that DNA-based

immunizations targeting model tumor antigens such as chicken ovalbumin (6), β-galactosidase (7), or CEA (8) induce protective immune responses leading to

rejection of a subsequent, normally lethal challenge with antigen-expressingtumor cells

Vaccines consisting of naked plasmid DNA have several potential tages over alternative immunization approaches relying on the delivery ofpurified or recombinant proteins, or live attenuated or recombinant viruses.They offer the promise of a readily deliverable, molecularly defined reagent

advan-that results in antigen synthesis in situ, but advan-that is neither infectious nor capable

of replication Importantly, both humoral and cell-mediated immune responsesmay be elicited against multiple defined antigens simultaneously Furthermore,

it may become possible to manipulate the nature of the resulting immuneresponse through the co-delivery of genes encoding immunomodulatingcytokines or costimulatory molecules Genetic constructs can be modified,allowing for the removal or insertion of transmembrane domains, signalsequences, or other residues that affect the intracellular trafficking and subse-quent processing of antigen The sequence may also be modified by site-directed mutagenesis, permitting single amino-acid exchanges designed toenhance the antigenic potency of individual epitopes or to abolish unwantedphysiologic effects of the wild-type protein Importantly, plasmid DNA,encoding suitable antigens or immune modulators, can readily and economi-cally be constructed and produced in large quantities with a high degree ofpurity and stability

2 DNA Vaccines: Antigen Expression, Processing,

and Presentation

It is now well established that injection of plasmid DNA through variousmethods and routes can induce both humoral and cell-mediated immuneresponses Significant titers of neutralizing anti-viral antibodies and potentcytotoxic T lymphocyte (CTL) responses have been documented in a number

of experimental animal models (4,9) Importantly, antigen-specific antibodies

and CTL reactivity could be detected in rodents longer than 1 yr after

immuni-zation (9) These studies suggest that plasmid DNA immuniimmuni-zation may

pro-mote long-lasting humoral and cellular immune responses qualitatively similar

to live attenuated or recombinant viral vaccines without the safety hazards ofinoculation of live virus

2.1 Effects of Variations in Gene Delivery and Expression

Plasmid DNA immunization has been accomplished using epidermal,

mucosal, intramuscular, and intravenous routes of administration (3) Striated

muscle initially was considered to be the only tissue capable of efficiently

tak-ing up and expresstak-ing free plasmid DNA in aqueous solution (10–12) This in

vivo gene transfer method was extensively studied by gene therapists as asimple way to deliver recombinant proteins such as Factor IX, growth hor-mone, or α-1-antitrypsin Transfection of muscle fibers could readily be dem-

onstrated using reporter genes such as β-galactosidase or firefly luciferase.Indeed,β-galactosidase activity could be detected in up to 5% of myofibrilsand luciferase activity was demonstrated up to 19 mo following intramuscular

injection of plasmid (11) Analyses by PCR and Southern blotting revealed

that the injected plasmid persisted episomally, presumably due to the low over of myocytes in vivo The gene gun has been of particular interest as analternative to intramuscular injection as the mode of delivery of plasmid DNA.The gene gun propels DNA-coated gold particles directly into the cytoplasm ofcells in target tissue by means of electrical discharge or helium pulse

turn-(1,3,13,14) Expression of biolistically delivered DNA encoding idase or luciferase was found to be transient in bombarded epidermis or liver,

β-galactos-and low levels of long-term expression could be observed in the dermis (13).

Both direct injection of plasmid DNA in saline and particle-mediated ery of plasmid DNA have now been extensively investigated as immunizationmethods Particle-mediated delivery of DNA to the epidermis of mice usingthe gene gun required up to 5000 times less DNA than intramuscular or intrad-ermal inoculation of DNA in aqueous solutions for the induction of immune

deliv-responses (14) Following injection of plasmid DNA in saline, cells

presum-ably take up DNA from extracellular spaces In contrast, using the gene gun,DNA is delivered directly into the cell cytoplasm These differences in genedelivery are believed to account for the fact that efficient transfection andinduction of immune responses can reproducibly be achieved with nanogramquantities of plasmid DNA using the gene gun, whereas injection of plasmidDNA in saline usually requires 25–100 µg (15,16) It is important to note, how-

ever, that the transfection efficiency of the target tissue does not necessarilycorrelate with the efficiency of immunization Indeed, one of the striking re-sults of early studies examining different routes of DNA delivery was that in-tradermal injection of free plasmid DNA can elicit potent immune responses tothe encoded antigen despite the relatively low efficiency of DNA uptake and

expression in skin when compared to muscle (17).

Recent evidence suggests that the site and method of DNA delivery mayaffect the nature of the immune response induced against antigens encoded by

plasmid DNA (16,18) The two most common targets for gene delivery, skin

and muscle, are generally considered to have significant differences inimmunocompetency The skin and mucous membranes are the site where mostforeign antigens are normally encountered, and these tissues have highlydeveloped immune surveillance functions The epidermis contains numerousbone-marrow-derived Langerhans cells (LC), which are professional antigen

presenting cells specialized for the initiation of immune responses (19)

Resi-dent LC can efficiently take up and process foreign antigens In addition, bothkeratinocytes and LC can secrete proinflammatory cytokines such as IL-1,